Platelet activation play critical roles in the development of thrombotic diseases such as heart attack and stroke. The roles of platelets in thrombosis involves two major aspects: Firstly, platelet adhesion, aggregation and secretion of granule contents are important in the development of atherosclerosis, which causes chronic vascular injury. Secondly, platelet activation at the site of vascular injury is critical in initiating arterial thrombosis. An intracellular secondary messenger molecule, cGMP, is synthesized during platelet activation. The role of cGMP in platelet activation has been controversial since the discovery of cGMP. It has been a prevailing concept in the past 30 years that cGMP, by activating the cGMP-dependent protein kinase (PKG), inhibits platelet activation. This concept, however, has been challenged by our recent finding that cGMPand PKG play a biphasic role in platelet activation, We show that cGMP plays a stimulatory role when low concentrations of cGMP is produced upon platelet agonist stimulation, but becomes inhibitory at high cGMP concentrations. During the last funding period, we have made significant progress in understanding the mechanisms of the biphasic roles of the cGMP pathway in platelet activation. We have found that the cGMP-PKG pathway plays a general role in stimulating platelet secretion and secretion-dependent platelet aggregation induced by most platelet agonists. We have obtained evidence supporting a new upstream signaling pathway of cGMP during platelet activation involving phosphoinositide 3-kinase, various forms of Akt and nitric oxide (NO) synthase. We have also obtained evidence supporting a novel downstreamsignaling mechanism of cGMP involving various isoforms of PKG and PKG-dependent sequentially activation of p38 and ERK mitogen-activated protein kinase pathways. These discoveries allow us to hypothesize a novel signaling pathwaythat stimulates platelet secretion. In this pathway, platelet agonists, by activating different isoforms of PI3K, Akt, NOS3,soluble guanylyl cyclase, and PKG, and via the p38 and ERK pathways, induce platelet secretion, which amplifies and stabilizes platelet aggregation. To test this hypothesis, we propose following specific aims: 1.To investigate the role of different Akt isoforms as upstream activators of the cGMP pathway during platelet activation. 2. To determine mechanisms of agonist-induced platelet NO production, its regulation, and its role in platelet secretion and aggregation. 3.To characterize the roles and the signaling mechanisms of different isoforms of PKGin platelet secretion and aggregation.